On-site Gram staining that increases a post-test probability of an ominous infection: a case of necrotizing fasciitis caused by Vibrio vulnificus: a case report

Background Gram staining is a classic but standard and essential procedure for the prompt selection of appropriate antibiotics in an emergency setting. Even in the era of sophisticated medicine with technically developed machinery, it is not uncommon that a classic procedure such as Gram staining is the most efficient for assisting physicians in making therapeutic decisions in a timely fashion. Case presentation A 65-year-old Asian man with alcoholic cirrhosis complicated by esophageal varices was brought to the emergency division of Saga Medical School Hospital in early August, complaining of severe pain, redness, swelling, and purpura of the lower extremities. On physical examination he appeared in a critically ill condition suggestive of deep-seated soft tissue infection, raising a pre-test probability of streptococci, staphylococci, Vibrio sp., or Aeromonas sp. as a causative pathogen. A characteristic of his residency in an estuarine area is that raw seafood ingestion, as documented in this patient prior to the current admission, predisposes those who have a chronic liver disease to a life-threatening Vibrio vulnificus infection. Given the pathognomonic clinical features suggestive of necrotizing fasciitis, our immediate attempt was to narrow down the differential list of candidate pathogens by obtaining clinical specimens for microbiological investigation, thus inquiring about the post-test probability of the causative pathogen. The Gram stain of the small amount of discharge from the test incision of the affected lesion detected Gram-negative rods morphologically compatible with V. vulnificus. After two sets of blood culture, intravenous meropenem and minocycline were immediately administered before the patient underwent emergency surgical debridement. The next day, both blood culture and wound culture retrieved Gram-negative rods, which were subsequently identified as V. vulnificus by mass spectrometry, matrix-assisted laser desorption/ionization. The antibiotics were switched to intravenous ceftriaxone and minocycline. Conclusion The pre-test probability of V. vulnificus infection was further validated by on-site Gram staining in the emergency division. This case report highlights the significance of a classic procedure.


Background
Necrotizing soft tissue infection caused by Vibrio vulnificus is a disease with a high mortality rate [1]. We experienced a case of V. vulnificus soft tissue infection presenting with septic shock. On-site Gram staining of the wound discharge right after the patient's arrival resulted in the successful management of the severe community-acquired sepsis and was limb saving. We present a Gram staining-based treatment strategy for V. vulnificus necrotizing soft tissue infection.

Case presentation
A week after seasonal heavy rainfall in early August, a 65-year-old Asian man was brought to the emergency division of Saga Medical School Hospital complaining of severe pain, redness, swelling, and purpura of the lower extremities. One day before the onset of symptoms, the patient had eaten sushi purchased at a grocery store. The patient's past medical history was remarkable for alcoholic cirrhosis complicated by esophageal varices, chronic heart failure with atrial fibrillation, and gout. He usually received oral esomeprazole, febuxostat, furosemide, spironolactone, apixaban, brotizolam, L-isoleucine, L-leucine, L-valine, sodium ferrous citrate, and loxoprofen. He is a carpenter and reported that he had drunk 2.5 cups of sake daily. He had no specific family history. He lived alone but his sister lived close to his house.
On arrival, he was oriented [Glasgow Coma Scale (GCS 15)], heart rate was 126 beats per minute (bpm), blood pressure 82/53 mmHg, temperature 39.5 ℃, respiratory rate 22/min, and SpO 2 99% (O 2 2 L/min). His ocular conjunctival was icteric, cervical lymph nodes were not palpated. The heart and the lungs were clear to auscultation. The abdomen was slightly distended, but soft without tenderness. Bilateral lower legs showed redness, swelling, and purpura accompanied by severe pain on light touch, a constellation of clinical features together with underlying comorbidities suggestive of necrotizing soft tissue infection ( Fig. 1). His pupils were 3 mm/3 mm, the light reflex was brisk. He did not have quadriplegia. His white blood cell (WBC) count was 25,100/μL and C-reactive protein (CRP) 12.5 mg/dL. Thrombocytopenia and liver dysfunction were compatible with cirrhosis, and azotemia was noted ( Table 1). The sequential organ failure score (SOFA) [2] was 9 and the laboratory risk indicator for necrotizing fasciitis [3,4] was 8, strongly suggesting the patient was in septic shock due to necrotizing fasciitis. The small amount of discharge obtained by test incision of the affected thigh was subjected to on-site Gram-staining, in which numerous thick and curved, banana-shaped Gram-negative rods were observed (Fig. 2). Because of this characteristic microbiological morphology and the regional preponderance of Ariake Sea for V. vulnificuscontaminated seafoods including shellfish exclusively associated with a rainy season, a working diagnosis of V. vulnificus necrotizing fasciitis was considered probable. After two sets of blood cultures from both arms, the patient was put on intravenous meropenem at 3 g/ day intravenously, and minocycline at 200 mg/day intravenously within 15 minutes after arrival, after which he  The computed tomography (CT) scan showed that fatty plaques were opacified and soft tissue density was elevated in the anterior to the medial aspect of the bilateral lower legs, suggesting fasciitis. The next day, both blood cultures (aerobic and anaerobic) and wound cultures showed Gram-negative rods and were identified as V. vulnificus by mass spectrometry, matrix-assisted laser desorption/ionization (MALDI TOF-MS) [5]. The antibiotics were switched to intravenous ceftriaxone at 2 g/day intravenously and minocycline at 200 mg/day intravenously (Fig. 3). The patient's postsurgical course was uneventful being put on oral minocycline at 200 mg/day alone until day 30 when rupture of esophageal varices developed resulting in death 10 days later due to decompensated liver failure. Because his family did not wish to, an autopsy was not performed .

Discussion
We reported a case in which V. vulnificus was instantly suspected by Gram staining, and definitive treatment with meropenem plus minocycline, could be started within 15 minutes after the patient's arrival on the first day of admission. Gram staining is said to be important [6], but there are few studies describing practical strategies. V. vulnificus is a Gram-negative bacterium that causes fatal bacteremia, typically complicated by necrotizing soft tissue infections [1]. Patients with chronic liver disease or hemochromatosis are at high risk for severe disease [7]. The presence of chronic medical illness and the use of steroids may worsen the prognosis of necrotizing fasciitis [8]. The natural habitat of V. vulnificus is brackish water and it proliferates with constant salinity and water temperature: V. vulnificus grows in estuaries where salinity has optimally decreased due to flooding [9], and it has been suggested that non-cholera Vibrio infections may increase in higher latitudes due to recent increases in the sea water temperatures [10]. The patient developed deep-seated skin/soft tissue infection in his lower legs the day after eating raw fish caught near the mouth of the river, several days after a heavy rain and flood disaster.
Given these clinical and regional epidemiological features, including temporal weather conditions of heavy rain, pointing to V. vulnificus necrotizing fasciitis serving as a high pre-test probability of this ominous infection, There have been many previous reports that have recommended appropriate antibiotic regimens for V. vulnificus infection, including minocycline and ceftriaxone, or fluoroquinolones [11]. The importance of appropriate antimicrobial selection from the time of initial therapy has been emphasized in the Surviving Sepsis Campaign [12].
Rather than discussing target antimicrobial therapy following the identification of bacteria, trying to predict the most probable causative organism based on the theoretical assumption making use of fundamental clinical reasoning such as pre-or pro-test probability is not only fast and frugal, but crucially important for timely initiation of quasi-target therapy.
Although the clinical outcome of this case was poor, this does not contradict the usefulness of on-site Gramstaining in semi-identifying micro-organisms, which should continually be encouraged among emergency care physicians. This strategy will also contribute to the judicious use of antibiotics in the era of antimicrobial resistance.

Conclusion
Necrotizing fasciitis caused by V. vulnificus is fatal and requires early appropriate antimicrobial therapy. Estimation of the causative organism by Gram staining is of vital importance in the treatment strategy for this life-threatening disease.